PCR for MULTIPLE mutagenesis

So I have 40bp primers which have 2 mutations in them. In the first mutation, 2 nucleotides have to change. In the second mutation, only 1 has to change. I've been doing this for weeks, and have only been getting the second mutation when I get the sequencing results.

I'm doing 20 cycles in my PCR, I just raised the annealing temp to 68 degrees from 55 degrees, and the extension time is long enough.

I'm also now using the DNA with the second mutation as a template so that hopefully I can get that first mutation.

After, I DPN1 for usually 3 hours before transforming. And when I transform, sometimes I get just a few colonies that are just the template, and sometimes I get nothing.

Should I be increasing cycles? Or something else? Any suggestions would be helpful. Also, I'm using the Stratagene Site Directed Mutagenesis PCR kit, and it's new. And the DNTP has been aliquoted into small tubes so I'm not thawing and refreezing.

A 40 bp primer is long, and it could possibly promote secondary-structure formation (i.e. stalling Pfu poly). I'm having a similar problem with my reactions, but I think it's because the enzyme doesn't work anymore. Are you sure your Tm's are high enough? My boss recommended you take out 5 uL of PCR rxn and mix it with 5 uL of Pfu Turbo DNA Polymerase Buffer, 39 uL dd H20 and 1 uL DpnI (I've also done the same but used NEBuffer 4 instead). This cuts down the amount of template that DpnI has to chew up. Perhaps you could try that....oh, and can I borrow some of your Pfu Poly?!

I'm doing 20 cycles in my PCR, I just raised the annealing temp to 68 degrees from 55 degrees, and the extension time is long enough.

You could definitely increase PCR cycles from 20 to 30 -35. But, could you please post your rationale for increasing the annealing temp by such a difference?

I'm going to try increasing the cycles to that high, though my PI doesn't think it'll make a difference. The increase in annealing temperature was found on a troubleshooting site, but now I see I should just have increased the temp by increments of 2 degrees.

I performed transformation yesterday on the DPN1'd PCR products and got nothing today. I'm running out of ideas, hpefully increasing the cycle numbers helps.

A 40 bp primer is long, and it could possibly promote secondary-structure formation (i.e. stalling Pfu poly). I'm having a similar problem with my reactions, but I think it's because the enzyme doesn't work anymore. Are you sure your Tm's are high enough? My boss recommended you take out 5 uL of PCR rxn and mix it with 5 uL of Pfu Turbo DNA Polymerase Buffer, 39 uL dd H20 and 1 uL DpnI (I've also done the same but used NEBuffer 4 instead). This cuts down the amount of template that DpnI has to chew up. Perhaps you could try that....oh, and can I borrow some of your Pfu Poly?!

The Tm's of the primers are similar to Tm's of primers I've used before and have worked, so I'm not sure about that. I'm going to try that DPN1 procedure with the PCR products tomorrow as well to see what happens. I've been trying to get these mutations for like a month and a half now and they are too close together in the sequence to have separate primers.